Methods for increasing vascular density and maintaining viability of microvascular endothelial cells using trk receptor ligands

ABSTRACT

The present invention relates to methods of inducing or inhibiting the angiogenic process and promoting vessel growth or stabilization in an organ by modulating the trk receptor pathway. The present invention also relates to a method for treating a pathological disorder in a patient which includes administering a trk receptor ligand or an inhibitor or expression or activity of a trk receptor ligand. The present invention also relates to a method of screening for a modulator of angiogenesis, vessel growth, or vessel stabilization. Another aspect of the present invention is a method of diagnosing or monitoring a pathological disorder in a patient which includes determining the presence or amount of a trk receptor ligand or activation of a trk receptor ligand in a biological sample.

The present application is a divisional of U.S. patent Ser. No.11/589,659, filed Oct. 30, 2006, which is a continuation of U.S. patentapplication Ser. No. 09/830,520, filed Jul. 20, 2001, which is a 371 ofPCT/US99/25365, filed Oct. 28, 1999, all of which are herebyincorporated by reference and claims the benefit of U.S. ProvisionalPatent Application Ser. No. 60/105,928, filed Oct. 28, 1998 and U.S.Provisional Patent Application Ser. No. 60/119,994, filed Feb. 12, 1999,all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the use of trk receptor ligands inmethods for regulating angiogenesis and vascular integrity, such asmethods of inducing angiogenesis, promoting vessel growth orstabilization, treating pathological disorders, inhibiting angiogenesis,and diagnosing or monitoring a pathological disorder. The presentinvention also relates to a method of screening for a modulator ofangiogenesis, vessel growth, or vessel stabilization.

BACKGROUND OF THE INVENTION

Angiogenesis is a precisely regulated process which coordinates theassembly and differentiation of numerous cell types to form thearteries, capillaries and veins of the pre-existing vascular bed. Theprimitive vasculature is composed of an endothelial plexus, whichrequire the recruitment of pericytes and vascular smooth muscle cells bysoluble growth factors secreted by endothelial cells to pattern thevessels into arteries and veins (Risau, “Mechanisms of Angiogenesis,”Nature 386:671-674 (1997)). In the final steps of vessel formation, thenewly formed endothelial cells are stabilized by the extracellularmatrix, the formation of a basement membrane and ensheathment withpericytes and smooth muscle cells. Numerous polypeptide growth factorshave been implicated in initiating vasculogenesis and angiogenicsprouting, including fibroblast growth factors (bFGF and FGF-2),vascular endothelial growth factor (VEGF), and the angiopoietins(Darland et al., “Blood Vessel Maturation: Vascular Development Comes ofAge,” J. Clin. Invest. 103:167-168 (1999); Ferrara et al., “The Biologyof Vascular Endothelial Growth Factor,” Endocrin. Rev. 18:4-25 (1997)).In addition, platelet derived growth factor B (PDGF-BB), angiopoietin-1(ang-1), ephrin B2, and TGFβ have been shown to regulate later aspectsof the angiogenesis process, in the recruitment of mural cells, and inthe patterning of the vascular bed (Yancopoulos et al., “Vasculogenesis,Angiogenesis and Growth Factors: Ephrins Enter the Fray at the Border,”Cell 93:661-664 (1998); Lindahl et al., “Pericyte Loss and MicroaneurysmFormation in the PDGF-B-deficient mice,” Science 277:242-245 (1997);Dickman et al., “Defective Haematopoiesis and Vasculogenesis inTransforming Growth Factor Beta 1 Knock Out Mice,” Development121:1845-1854 (1995); Yang et al., “Angiogenesis Defects and MesenchymalApoptosis in Mice Lacking SMAD5,” Development 126:1571-1580 (1999)).Very little is known about growth factors which regulate thestabilization and survival of the mature vasculature, althoughangiopoietin-1 has been proposed as a candidate molecule. Of thesefactors, only VEGF has been rigorously tested for its ability toinitiate angiogenesis in adults in preclinical and clinical trials(Ferrara et al., “The Biology of Vascular Endothelial Growth Factor,”Endocrin. Rev. 18:4-25 (1997); Mack et al., “Biologic Bypass With theUse of Adenovirus-Mediated Gene Transfer of the ComplementaryDeoxyribonucleic Acid for Vascular Endothelial Growth Factor 121Improves Myocardial Perfusion and Function in the Ischemic PorcineHeart,” J. Thoracic and Cardiovascular Surgery, 115:168-176 (1998);Losordo et al., “Gene Therapy for Myocardial Angiogenesis: InitialClinical Results with Direct Myocardial Injection of phVEGF165 as SoleTherapy for Myocardial Ischemia,” Circulation 98:2800-2804 (1998)).Although delivery of VEGF by gene transfer can induce an angiogenicresponse in ischemic tissues, exogenous VEGF induces the formation offragile, dilated and malformed vessels (Springer et al., “VEGF GeneDelivery to Muscle: Potential Role for Vasculogenesis in Adults,”Molecular Cell 2:549-558 (1998); Drake et al., “Exogenous VascularEndothelial Growth Factor Induces Malformed and Hyperfused VesselsDuring Embryonic Development,” Proc. Natl. Acad. Sci. 92:7657-7661(1995)). In addition, recent studies suggest that the endothelial cellsof postnatal vessels may become independent of VEGF for their continuedsurvival within several weeks of birth in rodents (Gerber et al., “VEGFis Required for Growth and Survival in Neonatal Mice,” Development126:1149-1159 (1999)). Thus, the ultimate endpoint is the definition ofthe cellular steps and molecular sequences that direct and maintainmicrovascular assembly leading to therapeutic targets for repair andadaptive remodeling.

In recent studies, the roles of the neurotrophins in regulatingcardiovascular development and modulating the vascular response toinjury have been investigated (Donovan et al., “Neurotrophin-3 isRequired for Mammalian Cardiac Development: Identification of anEssential Nonneuronal Neurotrophin Function,” Nature Genetics 14:210-213(1996); Donovan et al., “Neurotrophin and Neurotrophin Receptors inVascular Smooth Muscle Cells: Regulation of Expression in Response toInjury,” A. J. Path. 147:309-324 (1995); Kraemer et al., “NGF ActivatesSimilar Intracellular Signaling Pathways in Vascular Smooth Muscle Cellsas PDGF-BB But Elicits Different Biological Responses,” Arteriol.Thromb. And Vasc. Biol. 19:1041-1050 (1999)). The neurotrophins todayconsist of a family of five related polypeptide growth factors: nervegrowth factor (NGF), brain derived neurotrophic factor (BDNF), andneurotrophins 3, 4 (also referred to as neurotrophin 5), and 6 (NT-3,NT-4, NT-6) (Lewin et al., “Physiology of the Neurotrophins,” Ann. Rev.Neuro. 19:289-317 (1996)). These structurally related proteins mediatetheir actions on responsive neurons by binding to two classes of cellsurface receptor (Lewin et al., “Physiology of the Neurotrophins,” Ann.Rev. Neuro. 19:289-317 (1996)). The low affinity neurotrophin receptor,p75, binds all neurotrophins and modulates signaling initiated by thesecond class of neurotrophin receptors, the trk family of receptortyrosine kinases (what was originally identified as the trk tyrosinekinase receptor is now referred to as trk A, one member of the trkfamily of receptors). Trk A, trk B, and trk C tyrosine kinases serve asthe receptors for NGF, BDNF, and NT-3, respectively, and trk B can alsobe activated by NT-4.

NT-3 initiates a number of trophic effects on neurons expressing itsreceptor, trk C, ranging from mitogenesis, promotion of survival, ordifferentiation, depending on the developmental stage of the targetcells (Chalazonitis, “Neurotrophin-3 as an Essential Signal for theDeveloping Nervous System,” Molecular Neurobiology 12:29-53 (1996)). Thereported sites of action of NT-3 reside primarily in the peripheralnervous system (PNS), various areas of the central nervous system (CNS),and in the enteric nervous system (ENS). Id. Analyses of the phenotypesof transgenic mice lacking NT-3 or injection of embryos with a blockingantibody have revealed the essential role of NT-3 in development ofspecific populations of the PNS, and in particular of proprioceptive,nodose, and auditory sensory neurons and of sympathetic neurons. Id. Theactions of NT-3 also extend to modulation of transmitter release atseveral types of synapses in the periphery as well as in the adult CNS.Id.

NT-4 acts via the trk B receptor and supports survival of primarysomatic and visceral sensory neurons (Erickson et al., “Mice LackingBrain-Derived Neurotrophic Factor Exhibit Visceral Sensory Neuron LossesDistinct from Mice Lacking NT4 and Display a Severe DevelopmentalDeficit in Control of Breathing,” J. Neurosci. 16:5361-5371 (1996)). Themajor visceral sensory population, the nodose-petrosal ganglion complex(NPG), requires BDNF and NT-4 for survival of a full complement ofneurons, however, only one functional NT-4 allele is required to supportsurvival of all NT-4-dependent neurons. Id. NT-4 appears to have theunique requirement of binding to p75 for efficient signaling andretrograde transport in neurons (Ibáñez, “Neurotrophin-4: The Odd Oneout in the Neurotrophin Family,” Neurochemical Research 21:787-793(1996)). In addition, while all other neurotrophin knock-outs haveproven lethal during early postnatal development, mice deficient in NT-4have so far only shown minor cellular deficits and develop normally toadulthood.

Trk B receptors and BDNF are highly expressed by central and peripheralneurons, and gene ablation studies have demonstrated the critical roleof trk B and BDNF in neuronal differentiation and survival, with genetargeted animals exhibiting abnormalities in cerebellar function andrespiratory drive (Lewin et al., “Physiology of the neurotrophins,” Ann.Rev. Neuro. 19:289-317 (1996); Jones et al., “Targeted Disruption of theBDNF Gene Perturbs Brain and Sensory Neuron Development But Not MotorNeuron Development,” Cell 76:989-999 (1994); Erickson et al., “MiceLacking Brain-Derived Neurotrophic Factor Exhibit Visceral SensoryNeuron Losses Distinct From Mice Lacking NT4 and Display a SevereDevelopmental Deficit in Control of Breathing,” J. Neurosci.16:5361-5371 (1996); Schwartz et al., “Abnormal Cerebellar Developmentand Foliation in the BDNF (−/−) Mice Reveals a Role for Neurotrophins inCNS Patterning,” Neuron 19:269-281 (1997)).

However, the BDNF:trk B receptor system is expressed at high levels innonneuronal tissues, including muscle, lung, kidney, heart and thevasculature, where its biological functions are unclear (Donovan et al.,“Neurotrophin and Neurotrophin Receptors in Vascular Smooth MuscleCells: Regulation of Expression in Response to Injury,” A. J. Path.147:309-324 (1995); Timmusk et al., “Widespread and DevelopmentallyRegulated Expression of Neurotrophin-4 mRNA in Rat Brain and PeripheralTissues,” Eur. J. Neurosci. 5:605-613 (1993); Hiltunen et al.,“Expression of mRNAs for Neurotrophins and Their Receptors in DevelopingRat Heart,” Circ. Res. 79:930-939 (1996); Scarisbrick et al.,“Coexpression of the mRNAs for NGF, BDNF and NT-3 in the CardiovascularSystem of Pre- and Post-Natal Rat,” J. Neurosci. 13:875-893 (1993)).Prior studies have identified roles for the related neurotrophin, NT-3,and its receptor, trk C, in regulating cardiac septation andvalvulogenesis (Donovan et al., “Neurotrophin-3 is Required forMammalian Cardiac Development: Identification of an EssentialNonneuronal Neurotrophin Function,” Nature Genetics 14:210-213 (1996);Tessarollo et al., “Targeted Deletion of all Isoforms of the trk C GeneSuggests the Use of Alternate Receptor by its Ligand Neurotrophin-3 inNeural Development and Implicates trk C in Normal Cardiogenesis,” Proc.Natl. Acad. Sci. USA 94:14766-014781 (1997). In addition, it has beendemonstrated that BDNF and trk B are expressed by vascular smooth musclecells of the adult aorta, and expression of this ligand:receptor systemis upregulated in neointimal cells following vascular injury (Donovan etal., “Neurotrophin-3 is Required for Mammalian Cardiac Development:Identification of an Essential Nonneuronal neurotrophin Function,”Nature Genetics 14:210-213 (1996)). However, the biological actions ofBDNF and related neurotrophins in cardiovascular function anddevelopment have not been assessed.

The present invention is directed to functions of the neurotrophins andthe trk receptor family related to vascular biology.

SUMMARY OF THE INVENTION

The present invention relates to a method of inducing angiogenesis whichincludes delivering a trk receptor ligand in an amount effective toinduce angiogenesis.

The present invention also relates to a method for treating apathological disorder in a patient which includes administering a trkreceptor ligand in an amount effective to treat the pathologicaldisorder by inducing angiogenesis.

Another aspect of the present invention is a method of promoting vesselgrowth or stabilization which includes delivering a trk receptor ligandin an amount effective to promote vessel growth or stabilization.

Yet another aspect of the present invention is a method for treating apathological disorder in a patient which includes administering a trkreceptor ligand in an amount effective to treat the pathologicaldisorder by promoting vessel growth or stabilization.

The present invention also relates to a method of inhibitingangiogenesis which includes delivering an inhibitor of expression oractivity of a trk receptor ligand in an amount effective to inhibitangiogenesis.

The present invention also relates to a method for treating apathological disorder in a patient which includes administering aninhibitor of expression or activity of a trk receptor ligand in anamount effective to treat the pathological disorder by inhibitingangiogenesis.

The present invention further relates to a method of screening for amodulator of angiogenesis, vessel growth, or vessel stabilizationincluding providing a candidate compound and detecting modulation of atrk receptor ligand induced signal transduction pathway in a cell in thepresence of the candidate compound, wherein modulation of the signaltransduction pathway indicates that the candidate compound is amodulator of angiogenesis, vessel growth, or vessel stabilization.

Another aspect of the present invention is a method of diagnosing ormonitoring a pathological disorder in a patient which includesdetermining the presence or amount of a trk receptor ligand oractivation of a trk receptor ligand in a biological sample.

Although several growth factors have been identified as playing roles inthe initiation of angiogenesis, most notably VEGF, the present inventionshows that trk receptor ligands, e.g., trk B and trk C ligands, haveunique functions in vascular biology, including induction ofangiogenesis, vessel growth, and vessel stabilization. Unlike VEGF andVEGF receptors, which are expressed at high levels during embryogenesisbut are expressed at only low levels during adulthood, expression of thetrk B and trk C ligands by the vasculature is initiated during lategestation, and expression increases with postnatal life into adulthood.These distinctive patterns of expression suggest that endothelial cellsmay not require continued exposure to VEGF during adulthood, a pointrecently confirmed in animal models (Gerber et al., “VEGF is Requiredfor Growth and Survival in Neonatal Mice,” Development 126:1149-1159(1999), which is hereby incorporated by reference).

The in vitro and in vivo studies of the present invention support asurvival role for the trk B and trk C ligands, as opposed to the wellcharacterized mitogenic effects of VEGF on endothelial cells. As such,the trk B and trk C ligands demonstrate a critical stabilizing functionfor the vasculature, in preventing endothelial cell apoptosis. It isalso important to recognize the delivery of other angiogenic factors,like VEGF, at high levels has been accompanied by significant adverseeffects, with enhanced vessel fragility and the local induction ofhemangiomas, effects which might reflect the known mitogenic andpermeability promoting effects of VEGF (Drake et al., “ExogenousVascular Endothelial Growth Factor Induced Malformed and HyperfusedVessels During Embryonic Development,” Proc. Natl. Acad. Sci. USA92:7657-7661 (1995); Springer et al., “VEGF Gene Delivery to Muscle:Potential Role for Vasculogenesis in Adults,” Molecular Cell 2:549-558(1998), which are hereby incorporated by reference). In contrast,overexpression of a trk receptor ligand in the developing heart resultsin an increased capillary network, but no evidence of vascular fragilityor altered vessel permeability.

The actions of the trk receptor ligands also are distinguishable fromthose reported for the angiopoietins. Angiopoietins play a role inangiogenesis by conveying signals that stabilize the endothelial cellswithin newly formed blood vessels. In vitro studies suggest thatangiopoietin-1 may act as a survival factor for endothelial cells (Hayeset al., “Angiopoietin-1 and Its Receptor Tie-2 Participate in theRegulation of Capillary-Like Tubule Formation and Survival ofEndothelial Cells,” Microvasc. Res. 58:224-237 (1999), which is herebyincorporated by reference). As such, angiopoietin-1 is widely expressedby the smooth muscle cells surrounding endothelial cells, which expressthe angiopoietin-1 receptor, Tie2. Thus, unlike the trk receptor ligandswhich can act in an autocrine manner to support endothelial cellsurvival, angiopoietin-1 is produced by smooth muscle cells and acts ina paracrine manner to promote endothelial cell survival. Although bothangiopoietin-1 and the trk receptor ligands are expressed by cells ofthe postnatal and adult vasculature, the phenotype of BDNF null mutantand angiopoietin-1 or Tie2 null mutant animals is distinctive. There arealso important differences in the ability of trk receptor ligands andthe angiopoietins to initiate angiogenesis in in vivo models. In most invivo studies when angiopoietin-1 alone has been injected locally orsystemically into mice. Results have shown marginal changes inangiogenesis. In contrast, trk receptor ligands BDNF, NT-3 and NT-4appear to be similarly effective as VEGF in promoting the development ofvascular networks.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-J show the expression of neurotrophins in adult and embryonicrodent hearts. In particular, these figures show immunohistochemicaldetection of BDNF (panels A, and D), NT-3 (panel B), NT-4 (panel C), orkinase active trk B (panel E) in rodent hearts. Panels A, B, and C showsections of the left ventricular wall of adult female rats (6 weeks).Comparable results were obtained with adult mouse heart sections. PanelsD and E show sections of the ventricular wall of BDNF (+/+) mouseembryos at E18.5. Panel F shows heart sections from a BDNF (+/+) E18.5embryo incubated with anti-BDNF antisera to document antiseraspecificity. In panels A-F, VIP-based immunodetection was utilized,yielding a red reaction product. Panels G, H, I, and J show doubleimmunofluorescence detection of PECAM (CD31) reactivity (using arhodamine-conjugated primary antibody) and either BDNF reactivity(panels G and I) or kinase active trk B reactivity (using FITCconjugated secondary antibody) (panels H and I), with sections of adultmouse hearts (panels G and H) or E18.5 mouse hearts (panels I and J).Bars, 50 μm (panels A, B, and C); 25 μm (panels D, E, and F); 30 μm(panels G, H, I, and J).

FIGS. 2A-H show that BDNF (−/−) animals exhibit hemorrhage within theventricular walls through histological analyses of hearts of animalssacrificed within 8 hours of birth. Panels A, B, C, and D are sectionsstained with hematoxylin and eosin. ra and la=right and left atria; ryand lv=right and left ventricles; asd=atrial septal defect. Panels E, F,G, and H show thin sections of Epon embedded, toluidine blue stainedtissues from P0 animals. Note the abnormal arteriole, but normal venulein the BDNF (−/−) sections. Panels A, C, E, and G are BDNF (+/+)animals. Panels B, D, F, and H are BDNF (−/−) animals. Bars, 150 μm(panels A and B); 50 μm (panels D, E, and F); 5 μm (panels G and H).

FIGS. 3A-G show endothelial cell abnormalities in BDNF (−/−) animals.Panels A, B, and C are electron microscopic analyses of the leftventricular wall from BDNF (+/+) (panel A) or (−/−) (panels B and C) P0littermates. Vacuolated endothelial cells with an extensive anddisorganized extracellular matrix were consistently detected in thecapillaries and arterioles of the BDNF (−/−) animals. Panels D, E, F,and G show the use of immunofluorescence microscopy to detect PECAM(CD31) (rhodamine) and TUNEL (FITC) positivity in E18.5 embryos (panelsD and E) or P2 neonates (panels F and G). Panels D and F are sectionsfrom BDNF (+/+) animals, and panels E and G are sections from BDNF (−/−)animals. c=cardiac myocyte; en=endothelial cell; e=perivascular edema.Bars, 0.4 μm (panels A and B); 0.2 μm (panel C); 30 μm (panels D, E, F,and G).

FIGS. 4A-F show intramyocardial hemorrhage and vascularization in BDNF(−/−) embryos. E16.5 embryos (panels A and B) and E17.5 embryos (panelsC, D, E, and F) were harvested from BDNF (+/−) females, and sections ofthe developing heart were analyzed histologically following hematoxylinand eosin staining Panels E and F show immunohistochemical analysis ofintramyocardial vessels using an anti-CD31 antisera to detectendothelial cells within arterioles, venules, and capillaries of theventricular wall. Panels A, C, and E are BDNF (+/+) animals. Panels B,D, and F are BDNF (−/−) animals. Bars, 40 μm (panels A, B, C, and D); 20μm (panels E and F).

FIGS. 5A-J show atrial septal formation in BDNF (+/+) and (−/−) embryosthrough histological and immunohistochemical analyses of atrial septalformation in BDNF animals at E11.5 (panels A and D), E14.5 (panels B andE), E16.5 (panels C and F), and P0 (panels G and H). Sections of theatrial septum were stained with hematoxylin and eosin in BDNF (+/+)animals (panels A, B, and C) and BDNF (−/−) animals (panels D, E, andF). p=septum primum; s=septum secundum. Panels G, H, I, and J show theuse of immunohistochemical analysis to detect CD31 (panel H), BDNF(panel I), and kinase active trk B (panel J) to assess expression in theregion of the atrial septum of E18.5 BDNF (+/+) embryos. Preincubationof trk B specific antisera with the immunizing peptide confirms antiseraspecificity on sections from E18.5 BDNF (+/+) embryos (panel G). Bars,40 μm (panels A-F); 20 μm (panels G-J).

FIGS. 6A-G show that BDNF supports the survival of cardiac microvascularendothelial cells. Panels A and B show flow cytometric analysis ofcardiac microvascular endothelial cells incubated with anti-CD31antisera (panel A) or control IgG (panel B). 97% of cells exhibit CD31reactivity and 1% react with control IgG. Panel C shows RT-PCR analysisof transcripts for BDNF and kinase active trk B mRNA in cardiacmicrovascular endothelial cells (“ECs”) and adult murine brain (B).Amplification of BDNF (360 bp) and the regions of the kinase domain oftrk B (571 bp) are detectable in cardiac endothelial cells and adultbrain samples. To ensure the absence of DNA contamination, RNA sampleswere amplified using primers without the reverse transcription step andthese reactions yielded no products (no RT). Panels D, E, and F showTUNEL analysis of microvascular endothelial cells. ECs were cultured inmedia containing 10% serum (panel D), or in media containing 0% serum(panels E and F) in the presence of BDNF (100 ng/ml) (panel F) for 48hours. 1500 cells per sample were analyzed and the mean and standarddeviation of four samples is indicated. Results are representative oftwo experiments performed in quadruplicate on cultures from differentlitters of animals (Magnification: 20×). Panel G shows flow cytometricanalysis of annexin V binding. Cardiac microvascular endothelial cellswere cultured in the indicated conditions in the presence of BDNF (25ng/ml) or VEGF (10 ng/ml) for 48 hours prior to incubation withFITC-annexin V and propidium iodide for flow cytometry of 1×10⁴ cellsper condition. Results are representative of two independent experimentsperformed on cultures from different litters of animals.

FIGS. 7A-L show that overexpression of BDNF in gestational heartsresults in increased capillary density through the analysis of Nes-BDNFhearts from E18.5 embryos (panels A, C, D, G, I, and K) or wildtypelittermates (panels B, E, F, H, J, and L). Panels A and B show thathistologic analysis reveals abnormal vascularity of Nes-BDNF ventricularwall. Panels C, D, E, and F show that immunofluorescence detection ofBDNF demonstrates increased expression of Nes-BDNF (panel F) as comparedwith wildtype littermates (panel D). Preincubation of the BDNF antiserawith the immunizing peptide prior to immunofluorescence (panels C and E)confirms the specificity of the antisera. Immunofluorescence on pairedsamples was performed in parallel, using FITC-conjugated secondaryantisera and imaged by optical sectioning at identical settings. Resultsare representative of those observed with three transgenic and threewildtype embryos. Panels E and F show that CD31 immunoreactivity revealsan enhanced vessel density in Nes-BDNF embryos as compared to wildtypelittermates. Immunoreactivity was detected using a VIP substrate (redreaction product). Tissue sections are representative of those analyzedin four transgenic and four wildtype littermates. Panels G and H showthat α-actinin immunoreactivity, to detect vascular smooth muscle cells,is similar in Nes-BDNF transgenic and wild-type hearts. Note positivityof the large vessel in the wildtype section, but absence of reactivityof capillaries in wildtype and Nes-BDNF embryos. Panels I and J showPCNA immunoreactivity in sections of wildtype and Nes-BDNF embryonichearts. Panels G-H show results that are representative of thoseanalyzed in four transgenic and four wildtype littermates. Bars, 15 μm(panels A and B); 100 μm (panels C-F); 40 μm (panels G-L).

FIGS. 8A-E show en bloc analysis of Matrigel containing recombinantgrowth factors. Matrigel containing 50 ng/ml of the indicated growthfactor, or with no growth factor addition (control), was injectedsubcutaneously in the region of the rectus abdominus of six week oldmice. After 14 days, animals were sacrificed, and the Matrigel plug wasvisualized following dissection of the anterior abdominal wall (10×magnification).

FIGS. 9A-D show histological analysis of Matrigel sections containingthe indicated growth factor (at 50 ng/ml) or no additional growth factor(control). Following paraformaldehyde fixation, paraffin embedded tissuewas sectional at 10 microns, and processed with hematoxylin and eosinstaining A minimum of 7 mm of tissue was analyzed in serial sectionanalysis and representative sections were photographed at the indicatedmagnifications (left panels: 40×; right panels: 160×).

FIGS. 10A-F show histological analysis of Matrigel sections containingthe indicated growth factor (at 50 ng/ml) or no additional growth factor(control). Following paraformaldehyde fixation, paraffin embedded tissuewas sectional at 10 microns, and processed with hematoxylin and eosinstaining A minimum of 7 mm of tissue was analyzed in serial sectionanalysis and representative sections were photographed at the indicatedmagnifications (left panels: 40×; right panels: 160×).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of inducing angiogenesis whichincludes delivering a trk receptor ligand in an amount effective toinduce angiogenesis.

In a preferred embodiment, the trk receptor ligand is a trk B receptorligand.

In another preferred embodiment, the trk receptor ligand is a trk Creceptor ligand.

As used herein, trk ligands include proteins or polypeptides andfragments thereof, including the native neurotrophins and mutantsthereof, small chemical molecules, recombinant molecules, and chimericmolecules which interact with and activate trk receptors. Chimeric trkreceptor ligands include mutagenized neurotrophins which are capable ofactivating more than one trk receptor (see, e.g., Urfer et al.,“Specificity Determinants in Neurotrophin-3 and Design of Nerve GrowthFactor-Based trkC Agonists by Changing Central Beta-Strand BundleResidues to Their Neurotrophin-3 Analogs,” Biochemistry 36:4775-4781(1997); Ilag et al., “Pan-Neurotrophin 1: A Genetically EngineeredNeurotrophic Factor Displaying Multiple Specificities in PeripheralNeurons in vitro and in vivo,” Proc. Natl. Acad. Sci. USA 92:607-611(1995), which are hereby incorporated by reference).

Suitable trk receptor ligands include brain derived neurotrophic factor(“BDNF”), NT-3, NT-4, and recombinant and small molecule mimics thereof.

BDNF is a neurotrophin best characterized for its survival anddifferentiative effects on neurons expressing the trk B receptor kinase.Deficient expression of BDNF does not affect the assembly or patterningof endothelial cells in intramyocardial vessels, but impairs theirsurvival. BDNF deficiency induces endothelial cell apoptosis, leading tointraventricular wall hemorrhage, depressed cardiac contractility, andearly postnatal death. In contrast, ectopic BDNF overexpression isassociated with increased capillary density and increased survival ofcardiac microvascular endothelial cells. Thus, expression of BDNF isrequired for the stabilization of intramyocardial vessels during lateembryogenesis, through direct actions on endothelial cells.

NT-3 is a member of the neurotrophin family and exhibits significanthomology with NGF and BDNF (Hohn et al., “Identification andCharacterization of a Novel Member of the Nerve GrowthFactor/Brain-Derived Neurotrophic Factor Family,” Nature 334:339-341(1990), which is hereby incorporated by reference). NT-3 mediates itsactions on trk C expressing neurons, and its role in promoting thesurvival of subclasses of sensory and sympathetic neurons during thedevelopment of the peripheral nervous system has been establishedthrough the analysis of gene targeted mice (Snider, “Functions of theNeurotrophins During Nervous System Development: What the Knockouts areTeaching Us,” Cell 77:627-638 (1994), which is hereby incorporated byreference). NT-3 is highly expressed by capillaries in adult rodentheart (see FIG. 1). In addition, NT-3 promotes angiogenesis in aMatrigel assay (see Example 14, below).

NT-4 is the most divergent member of the neurotrophins and, in contrastwith other neurotrophins, its expression is ubiquitous and lessinfluenced by environmental signals (Ibáñez, “Neurotrophin-4: The OddOne Out in the Neurotrophin Family,” Neurochemical Research, 21:787-793(1996), which is hereby incorporated by reference). It shares its tworeceptors (trkB and p75) with other members of the neurotrophin family,e.g., BDNF. Id. Evidence suggests that the level of NT-4 mRNA inskeletal muscle is controlled by muscle activity and that muscle derivedNT-4 is an activity dependent neurotrophic signal for growth andremodeling of adult motor neuron innervation, and may thus be partlyresponsible for the effects of exercise and electrical stimulation onneuromuscular performance. Id. NT-4 mRNA is expressed at significantlevels in the embryonic heart, but falls to undetectable levels in theadult heart (Timmusk et al., “Widespread and Developmentally RegulatedExpression of Neurotrophin-4 mRNA in Rat Brain and Peripheral Tissues,”Eur. J. Neurosci. 5:605-613 (1993), which is hereby incorporated byreference). However, NT-4 is as potent as VEGF in promoting theformation of vascular networks in an in vivo Matrigel assay (see Example14, below).

The trk receptor ligand may be employed in accordance with the presentinvention by administering a protein or polypeptide ligand.

The trk receptor ligand may also be employed in accordance with thepresent invention by expression of such trk receptor ligand in vivo,which is often referred to as “gene therapy.”

The genes encoding the trk receptor ligand and proteins or polypeptidesderived therefrom are known in the art, as well as methods for producingsuch proteins or polypeptides, e.g., recombinantly (U.S. Pat. No.5,180,820 to Barde et al.; U.S. Pat. No. 5,229,500 to Barde et al.; U.S.Pat. No. 5,438,121 to Barde et al.; U.S. Pat. No. 5,453,361 toYancopoulos et al.; U.S. Pat. No. 5,770,577 to Kinstler et al.; U.S.Pat. No. 5,235,043 to Collins et al.; Enfors et al., “Molecular Cloningand Neurotrophic Activities of a Protein with Structural Similarities toNerve Growth Factor: Developmental and Topographical Expression in theBrain,” Proc. Natl. Acad. Sci. USA 87:5454-5458 (1990); Hohn et al.,“Identification and Characterization of a Novel Member of the NerveGrowth Factor/Brain-Derived Neurotrophic Family,” Nature 344:339-341(1990); Jones et al., Molecular Cloning of a Human Gene that is a Memberof the Nerve Growth Factor Family,” Proc. Natl. Acad. Sci. USA87:8060-8064 (1990); Maisonpierre et al., “Neurotrophin-3: ANeurotrophic Factor Related to NGF and BDNF,” Science 24:1446-1451(1990); and Rosenthal et al., “Primary Structure and Biological Activityof a Novel Human Neurotrophic Factor,” Neuron 4:767-773 (1990); Fandl etal., “Characterization and Crystallization of Recombinant HumanNeurotrophin-4,” J. Biol. Chem. 269:755-759 (1994); Ibanez et al.,“Neurotrophin-4 is a Target-Derived Neurotrophic Factor For Neurons ofthe Trigeminal Ganglion,” Development 117:1345-1353 (1993); Ip et al.,“Mammalian Neurotrophin-4: Structure, Chromosomal Localization, TissueDistribution, and Receptor Specificity,” Proc. Natl. Acad, Sci. USA89:3060-3064 (1992); Hallbrook et al., “Evolutionary Studies of theNerve Growth Factor Family Reveal A Novel Member Abundantly Expressed inXenopus Ovary,” Neuron 6:845-858 (1991), which are hereby incorporatedby reference).

Cells may be engineered by procedures known in the art, including by useof a retroviral particle containing RNA encoding the trk receptor ligandof the present invention. Similarly, cells may be engineered in vivo forexpression of trk receptor ligand in vivo by procedures known in theart. As known in the art, for example, a producer cell comprising aretroviral particle containing RNA encoding the trk receptor ligand ofthe present invention may be administered to a patient for expression ofthe trk receptor ligand in vivo. These and other methods foradministering the trk receptor ligand of the present invention should beapparent to those skilled in the art from the teachings of the presentinvention.

Construction of appropriate expression vehicles and vectors for genetherapy applications will depend on the organ to be treated and thepurpose of the gene therapy. The selection of appropriate promoters andother regulatory DNA will proceed according to known principles, basedon a variety of known gene therapy techniques. For example, retroviralmediated gene transfer is a very effective method for gene therapy, assystems utilizing packaging defective viruses allow the production ofrecombinants which are infectious only once, thus avoiding theintroduction of wild-type virus into an organism. Alternativemethodologies for gene therapy include non-viral transfer methods, suchas calcium phosphate co-precipitation, mechanical techniques, forexample microinjection, membrane fusion-mediated transfer via liposomes,as well as direct DNA uptake and receptor-mediated DNA transfer.

Viral vectors which may be used to produce stable integration of geneticinformation into the host cell genome include adenoviruses, theadenoassociated virus vectors (AAV) (Flotte et al., Gene Ther., 2:29-37(1995); Zeitlin et al., Gene Ther., 2:623-31 (1995); Baudard et al.,Hum. Gene Ther., 7:1309-22 (1996); which are hereby incorporated byreference), and retroviruses. For a review of retrovirus vectors, seeAustin, Gene Ther., 1(Suppl 1):S6-9 (1994) and Eglitis, Blood, 71:717-22(1988), which are hereby incorporated by reference. Other viral vectorsare derived from herpesviruses, etc.

Retroviruses are RNA viruses which are useful for stably incorporatinggenetic information into the host cell genome. When they infect cells,their RNA genomes are converted to a DNA form (by the viral enzymereverse transcriptase). The viral DNA is efficiently integrated into thehost genome, where it permanently resides, replicating along with hostDNA at each cell division. This integrated provirus steadily producesviral RNA from a strong promoter located at the end of the genome (in asequence called the long terminal repeat or LTR). This viral RNA servesboth as mRNA for the production of viral proteins and as genomic RNA fornew viruses. Viruses are assembled in the cytoplasm and bud from thecell membrane, usually with little effect on the cell's health. Thus,the retrovirus genome becomes a permanent part of the host cell genome,and any foreign gene placed in a retrovirus ought to be expressed in thecells indefinitely.

Retroviruses are therefore attractive vectors, because they canpermanently express a foreign gene in cells. Moreover, they can infectvirtually every type of mammalian cell, making them exceptionallyversatile. In the design and use of retroviral vectors, the vectorsusually contain a selectable marker as well as the foreign gene to beexpressed. Most of the viral structural genes are gone, so these vectorscannot replicate as viruses on their own. To prepare virus stocks,cloned proviral DNA is transfected into a packaging cell. These cellsusually contain an integrated provirus with all its genes intact, butlacking the sequence recognized by the packaging apparatus. Thus, thepackaging provirus produces all the proteins required for packaging ofviral RNA into infectious virus particles but it cannot package its ownRNA. The packaging system may allow use of a variety of viral envelopesto alter viral tropism, and ability to infect human cells. Examplesinclude retroviral vectors using amphotropic, HIV-1/2, SIV, Gibbon ApeLeukemia Virus (“GALV”), or Vesicular Stomatis Virus (“VSV”) envelope.Vector packaging systems and/or backbones may be derived from varioussources such as MoMuLV, or even lentiviruses such as HIV-1, SIV, etc.RNA transcribed from the transfected vector is packaged into infectiousvirus particles and released from the cell. The resulting virus stock istermed helper-free, because it lacks wild-type replication-competentvirus. This virus stock can be used to infect a target cell culture. Therecombinant genome is efficiently introduced, reverse-transcribed intoDNA (by reverse transcriptase deposited in the virus by the packagingcells), and integrated into the genome. Thus, the cells now express thenew virally introduced gene, but they never produce any virus, becausethe recombinant virus genome lacks the necessary viral genes.

The invention, therefore, provides for the expression of trk receptorligands in vivo by methods including viral vectors which carry thenucleic acids encoding the trk receptor ligand.

Preferably, the trk receptor ligand is delivered in an assay system,sample, or target organ.

For inducing angiogenesis and promoting vascular survival, delivering aneffective amount of a trk receptor ligand includes delivering nanomolarconcentrations of ligand to the target site, as described foradministration of VEGF (see, e.g., Mack et al., “Biologic Bypass Withthe Use of Adenovirus-Mediated Gene Transfer of the ComplementaryDeoxyribonucleic Acid for Vascular Endothelial Growth Factor 121Improves Myocardial Perfusion and Function in the Ischemic PorcineHeart,” J. Thorac. Cardiovasc. Surg. 115:168-176 (1998); Magovern etal., “Direct in vivo gene Transfer to Canine Myocarium Using aReplication-Deficient Adenovirus Vector,” Ann. Thorac. Surg. 62:425-433(1996); Symes et al., “Gene Therapy with Vascular Endothelial GrowthFactor for Inoperable Coronary Artery Disease,” Ann. Thorac. Surg.68:830-836 (1999); Losordo et al., “Gene Therapy for MyocardialAngiogenesis,” Am. Heart J. 138(2, Pt. 2):132-141 (1999); Losordo etal., “Gene Therapy for Myocardial Angiogenesis: Initial Clinical ResultsWith Direct Myocardial Injection of phVEGF165 as Sole Therapy forMyocardial Ischemia,” Circulation 98:2800-2804 (1998), which are herebyincorporated by reference). For gene delivery, an effective amount issufficient quantities of the vector to ensure synthesis of nanomolarconcentrations of protein or polypeptide ligand in the target site.

In accordance with the method of the present invention, the trk receptorligand can be administered in vivo orally, intravenously,intramuscularly, intraperitoneally, subcutaneously, by intranasalinstillation, by application to mucous membranes, such as, that of thenose, throat, and bronchial tubes, intracerebrally, into cerebral spinalfluid, or by instillation into hollow organ walls or newly vascularizedblood vessels. It may be administered alone or with pharmaceutically orphysiologically acceptable carriers, excipients, or stabilizers, and canbe in solid or liquid form such as, tablets, capsules, powders,solutions, suspensions, or emulsions.

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing thetrk receptor ligand of the present invention and a carrier, for example,lubricants and inert fillers such as, lactose, sucrose, or cornstarch.In another embodiment, these compounds are tableted with conventionaltablet bases such as lactose, sucrose, or cornstarch in combination withbinders like acacia, cornstarch, or gelatin, disintegrating agents, suchas cornstarch, potato starch, or alginic acid, and a lubricant, likestearic acid or magnesium stearate.

The trk receptor ligand of the present invention may also beadministered in injectable dosages by solution or suspension of the trkreceptor ligand in a physiologically acceptable diluent with apharmaceutical carrier. Such carriers include sterile liquids, such aswater and oils, with or without the addition of a surfactant and otherpharmaceutically and physiologically acceptable carrier, includingadjuvants, excipients or stabilizers. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions.

For use as aerosols, the trk receptor ligand of the present invention insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The trk receptor ligand of the present invention also may beadministered in a non-pressurized form such as in a nebulizer oratomizer.

The present invention also relates to a method for treating apathological disorder in a patient which includes administering a trkreceptor ligand in an amount effective to treat the pathologicaldisorder by inducing angiogenesis in the manner described above.

In one embodiment, the pathological disorder is cardiac ischemia.Cardiac ischemia includes cerebrovascular disorders caused byinsufficient cerebral circulation. Thrombi or emboli due toatherosclerotic or other disorders (e.g., arteritis or rheumatic heartdisease) commonly cause ischemic arterial obstruction.

In another embodiment, the pathological disorder is a non-cardiacvascular disorder including atherosclerosis, renal vascular disease, andstroke.

In yet another embodiment, the pathological disorder is a wound. Suchwounds include, but are not limited to, chronic stasis ulcers, diabeticcomplications, complications of sickle cell disease, thallasemia andother disorders of hemoglobin, and post-surgical wounds.

In a further embodiment, the pathological disorder is a condition ofplacental insufficiency. Such conditions include, but are not limitedto, intrauterine growth retardation.

In yet a further embodiment, the pathological disorder unvascularizedtissue related to grafts and transplants (see, e.g., PCT InternationalApplication No, WO 99/06073 to Isner, which is hereby incorporated byreference).

Another aspect of the present invention is a method of promoting vesselgrowth or stabilization which includes delivering an effective amount ofa trk receptor ligand in an amount effective to promote vessel growth orstabilization in the manner described above.

Yet another aspect of the present invention is a method for treating apathological disorder in a patient which includes administering a trkreceptor ligand in an amount effective to treat the pathologicaldisorder by promoting vessel growth or stabilization in the mannerdescribed above.

In a preferred embodiment, the pathological disorder relates toendothelial cell apoptosis or necrosis. An example of such apathological disorder is vasculitis.

The present invention also relates to a method of inhibitingangiogenesis which includes delivering an inhibitor of expression oractivity of a trk receptor ligand in an amount effective to inhibitangiogenesis.

Suitable inhibitors include any component capable of blocking thebinding of ligands to the receptor, thus inhibiting receptor activation.

In one embodiment, the delivering includes delivering a nucleic acidsequence encoding an antisense molecule complementary to mRNA encoding atrk receptor ligand as is known in the art (antisense includesribozymes).

In another embodiment, the delivering includes delivering a receptorbody (Binder et al., “Selective Inhibition of Kindling Development byIntraventricular Administration of TrkB Receptor Body,” J. Neurosci.19:1424-1436 (1999), which is hereby incorporated by reference).Receptor bodies include the extracellular domain of the receptor and maybe bound to the Fc portion of an immunoglobulin molecule for delivery.Delivery of receptor bodies can be used to bind native trk receptorligand and thus prevent the activation of trk receptors.

For inhibition of angiogenesis, delivering an effective amount of aninhibitor of expression or activity of a trk receptor ligand includesdelivering sufficient inhibitor to inhibit nanomolar concentrations ofthe native ligand(s) in the target organ.

The present invention also relates to a method for treating apathological disorder in a patient which includes administering aninhibitor of expression or activity of a trk receptor ligand in anamount effective to treat the pathological disorder by inhibitingangiogenesis.

In one embodiment, the pathological disorder is a vascular proliferativedisease. Suitable vascular proliferative diseases include hemangiomasand proliferative retinopathy.

In another embodiment, the pathological disorder is cancer.

The present invention further relates to a method of screening for amodulator of angiogenesis, vessel growth, or vessel stabilization. Thismethod includes providing a candidate compound and detecting modulationof a trk receptor ligand induced signal transduction pathway in a cellin the presence of the candidate compound, wherein modulation of thesignal transduction pathway indicates that the candidate compound is amodulator of angiogenesis, vessel growth, or vessel stabilization.

In a preferred embodiment, the detecting comprises assessing trktyrosine phosphorylation. In particular, trk receptor activation can beassessed through the use of antibodies which specifically recognizetyrosine-phosphorylated epitopes in the cytoplasmic domain of activatedtrk receptors (Segal et al., “Differential Utilization of TrkAutophosphorylation Sites,” J. Biol. Chem. 271:20175-20181 (1996), whichis hereby incorporated by reference). As trk receptors become tyrosinephosphorylated following the binding of ligand, these reagents can beused to detect activated, but not inactive, trk receptors. Commercialsources of these reagents include Santa Cruz Biotechnologies, SantaCruz, Calif.

Another aspect of the present invention is a method of diagnosing ormonitoring a pathological disorder in a patient which includesdetermining the presence or amount of a trk receptor ligand oractivation of a trk receptor ligand in a biological sample.

Suitable pathological disorders include cardiac ischemia,atherosclerosis, renal vascular disease, stroke, a wound, placentalinsufficiency, unvascularized tissue related to grafts and transplants,disorders relating to endothelial cell apoptosis or necrosis,hemangiomas, proliferative retinopathy, and cancer.

In a preferred embodiment, the presence or amount of trk receptorligands in certain tissue, e.g., tumor cells, sclerotic vessels, andvascular channels surrounded by tumor cells, may be used as an earlymaker of tumor angiogenesis (Zagzag et al., “In Situ Expression ofAngiopoietins in Astrocytomas Identifies Angiopoietin-2 as an EarlyMarker of Tumor Angiogenesis,” Exp. Neurol. 159:391-400 (1999), which ishereby incorporated by reference).

Determining the presence or amount of a trk receptor ligand oractivation of a trk receptor ligand in a biological sample may beaccomplished using methods known to those of ordinary skill in the art.In one embodiment, the determining comprises assessing trk tyrosinephosphorylation, as described above.

Suitable biological samples include blood, urine, hair, cheek scrapings,semen, tissue biopsy, and saliva.

EXAMPLES Example 1 BDNF Mutant MICE

Heterozygous (+/−) BDNF mice (Ernfors et al., “Mice LackingBrain-Derived Neurotrophic Factor Develop with Sensory Defects,” Nature,368:147-150 (1994), which is hereby incorporated by reference), (STOCKBDNF^(tm1Jae) and C5713L/6J backcrossed BDNF^(tm1Jae)) were obtainedfrom Jackson Laboratories (Bar Harbor, Me.), and were intercrossed bybrother/sister matings for embryo analysis. The morning of the detectionof a vaginal plug was considered day 0.5, and the gestational age wasassigned. At the time of embryo harvest, morphologic criteria were usedin assigning developmental age. Key criteria included limb bud, eye andear development, crown-rump length and weight (Kaufman, “The Atlas ofMouse Development,” Academic Press, Inc. San Diego (1992), which ishereby incorporated by reference). The genotype of each embryo ornewborn mouse was determined by analysis of head derived DNA using PCRamplification with primer sequences as described in Ernfors et al.,“Mice Lacking Brain-Derived Neurotrophic Factor Develop with SensoryDeficits,” Nature 368:147-150 (1994), which is hereby incorporated byreference.

Mice were sacrificed and bodies fixed immediately in 3% paraformaldehydein phosphate buffered saline for 18 hours, and the contents of thethoracic cavity were dissected en bloc. Tissues were embedded inparaffin for histologic analysis. Tissues used for immunohistochemistrywere infiltrated with 30% sucrose prior to cryoprotection in 30%sucrose/OCT. The bodies of embryos of gestational age of E14.5 or lesswere embedded without prior dissection. Sections of 10 microns werestained using hematoxylin and eosin as described in Donovan et al.,“Neurotrophin-3 is Required for Mammalian Cardiac DevelopmentIdentification of an Essential Nonneuronal Neurotrophin Function,”Nature Genetics 14:210-213 (1996), which is hereby incorporated byreference). For electron microscopic analysis, the hearts wereimmediately removed from newborn mice sacrificed by decapitation, andfixed in Karnovsky's fixative (2% glutaraldehyde/paraformaldehyde incacodylate buffer) for 18 hours prior to embedding in Epon. Tissues weresectioned at 1 micron and stained with toluidine blue for initialevaluation and then ultrathin sections were cut with a diamond knife,counterstained with lead citrate, and viewed with an electronmicroscope.

Example 2 Immunohistochemical Analysis

Monoclonal antisera specific for α-actinin 1E12 (undiluted monoclonalsupernatant) was utilized for detection of vascular smooth muscle cells.Biotinylated PECAM-1 antibody specific for CD 31 (1:100 dilution, cloneMEC 13.3, Pharmingen, San Diego, Calif.) was used to detect endothelialcells and dc101 monoclonal antisera (Imclone, New York, N.Y.) was usedto detect flk-1. Polyclonal antisera specific for BDNF, NT-3, NT-4(sc-546, sc-547, sc-545 respectively, 1:50-1:500 dilution, Santa CruzImmunochemicals, Santa Cruz, Calif.) or kinase active trk B (sc-12-G,1:100 dilution, Santa Cruz Immunochemicals, Santa Cruz, Calif.) wereused on tissues which had been snap frozen over liquid nitrogen vaporand sectioned on a cryostat. The specificity of neurotrophin antiserahas been previously confirmed by the absence of staining of neuraltissues from the appropriate gene targeted mice. In addition,preincubation of polyclonal antisera with the immunizing peptide wasused to confirm antibody specificity. Sections were treated with 0.1%hydrogen peroxide prior to incubation with the primary antibody, andsignal amplification utilized the avidin:biotinylated horseradishperoxidase complex method (ABC Vectastain, Vector Labs, Burlingame,Calif.). TUNEL procedure was performed as per the manufacturer'srecommendation (BoeringerManheim, Chicago, Ill.) using frozen sections.Double immunofluorescence microscopy was performed using a ZeissAxioskop microscope (Thornwood, N.Y.), or a Zeiss confocal microscope(Thornwood, N.Y.) to generate 0.5 micron optical sections.

Example 3 Generation of NesPIXpBDNF Mice

The generation of transgenic mice has been described earlier (Ringstedtet al., “BDNF Regulates Reelin Expression and Cajal-Retzius CellDevelopment on the Cerebral Cortex,” Neuron 21:299-310 (1998), which ishereby incorporated by reference). Briefly, the Nes PIX-pBDNF constructconsisted of a region extending 5.8 kb upstream from the initiationcodon of the mouse nestin gene followed by a 1 kb fragment from thefifth exon of the mouse gene containing the complete BDNF protein codingsequence, a 300 bp long SV40 polyadenylation signal, and 5.4 kb of thenestin gene downstream sequence including introns 1, 2 and 3. Theconstruct was injected into fertilized mouse oocytes that weresubsequently transplanted into pseudopregnant females. Embryos wereharvested at E17.5-E18.5 from staged pregnant mothers, and weredecapitated and the thoracic contents were dissected and either fixed in4% paraformaldehyde prior to embedding in paraffin, or snap frozen overliquid nitrogen prior to frozen sectioning. Head tissue was used forgenotyping using PCR as described (Ringstedt et al., “BDNF RegulatesReelin Expression and Cajal-Retzius Cell Development on the CerebralCortex,” Neuron 21:299-310 (1998), which is hereby incorporated byreference).

Example 4 Capillary Counts

Immunohistochemistry was performed on heart sections using thebiotinylated anti-CD-31 antisera which detects all vascular endothelialcells (Gerber et al., “VEGF is Required for Growth and Survival inNeonatal Mice,” Development 126:1149-1159 (1999), which is herebyincorporated by reference). Immunostained sections were photographed at400×, and images were imported and analyzed using NIH Image.Subepicardial regions were randomly selected and the area ofimmunoreactivity was quantitated and expressed as a percent of the totalarea analyzed. Three independent fields were counted from each heartsection obtained, using tissue from three transgenic and three wildtypelittermates.

Example 5 Cell Culture

Microvascular endothelial cells were isolated from the hearts of C57/B1mice at postnatal day 2-4 according to established protocols (Lodge etal., “A Simple Method of Vascular Endothelial Cell Isolation,”Transplantation Proceedings 24:2816-2817 (1992), which is herebyincorporated by reference). In brief, minced hearts were digested withcollagenase and DNAse 1, and cells plated on gelatin coated plates.Endothelial cells were released by brief trypsinization after 48 hoursin culture, and maintained on gelatin coated plates in DMEM/F12 mediacontaining 5% fetal bovine serum, 0.1% mouse serum, insulin,transferrin, and selenium (1:100, Gibco, Rockville, Md.) and used atpassage 1 or 2. Using this procedure, approximately 0.5−1×10⁶ cells wereisolated from 20 neonatal hearts, and cell purity was quantitated usingacetylated LDL binding and CD-31 expression assessed by flow cytometricanalysis as described (Bergers et al., “Effects of AngiogenesisInhibitors on Multistage Carcinogenesis in Mice,” Science 284:808-812(1999), which is hereby incorporated by reference). For TUNEL analysis,4×10⁴ cells/cm² were plated on gelatin coated Permanox slides (NalgeneNunc, Naperville, Ill.)) and were cultured as above for 24 hours. Cellswere washed and re-fed with X-vivo containing 0% serum±the indicatedgrowth factor, or in X-vivo20 (Biowhittaker, Walkersville, Md.)containing 10% serum for 48 hours prior to fixation and TUNELassessment. 1500 cells in each well were scored for TUNEL positivity.For assessment of annexin V binding, cells were deeded onto gelatincoated 6 will plates and cultured as above with the addition of 1 nl/mlof bFGF to the media. After 24 hours, cells were washed, and re-fed withX-vivo20 containing 0% serum and additional growth factors as indicated,or X-vivo20 containing 10% serum. After 48 hours, cell suspensions weregenerated using PBS/EDTA, washed in serum free DMEM and annexin Vbinding was determined by incubating the cells with FITC-conjugatedAnnexin V (Immunotech, Miami, Fla.) in DME containing 1.5 mM Ca²⁺ on icefor ten minutes. After washing to remove unbound annexin V, cells wereincubated with propidium iodide and cell samples analyzed by flowcytometry using a Coulter Elite system (Miami, Fla.).

Example 6 RT-PCR

Total RNA was extracted from microvascular EC (passage 4-5) and fromadult murine brain as described (Chomczynski et al., “Single-Step Methodof RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-ChloroformExtraction,” Anal Biochem 162:156-159 (1987), which is herebyincorporated by reference). One microgram of total RNA was subjected toreverse transcription using murine leukemia virus transcriptase(Perkin-Elmer, Branchburg, N.J.). Total RNA not incubated with reversetranscriptase was used a negative control. RT-PCR was performed usingprimer sequences for BDNF or truncated trk B as described (Labouyrie etal., “Expression of Neurotrophins and their Receptors in Human BoneMarrow,” Am. J. Pathol. 154:405-415 (1999), which is hereby incorporatedby reference). Primer sequences for kinase active trk B were modifiedfrom Labourie et al., “Expression of Neurotrophins and their Receptorsin Human Bone Marrow,” Am. J. Pathol. 154:405-415 (1999), which ishereby incorporated by reference, to reflect codon usage in the murinesequence. The PCR products were resolved by electrophoresis in 7%acrylamide gels, followed by visualization with ethidium bromide.

Example 7 Echocardiographic Imaging

Within 48 hours of birth, all animals in a litter were subjected tosonographic imaging in a blinded fashion. Animals were imaged byplacement of a 40 MHz Scimed coronary probe (Boston Scientific Scimed,Minneapolis, Minn.) in warmed gel on the anterior chest wall, using aClear View Ultra Boston Scientific system (model 15006, Minneapolis,Minn.) with real time image analysis. Images of the short axis and longaxis of the heart were identified, and imaging proceeded for a minimumof four minutes per animal. The cardiac rate, chamber dimensions, andwall motion were determined on each animal by analysis of recordedimages, using the Diagnostic Off-line analysis system (Diagnostics,Inc., Houston, Tex.). The animals were sacrificed for genotyping andhistologic analysis within 4 hours of imaging.

Example 8 BDNF and trk B are Expressed by Vessels in Adult and EmbryonicRodent Heart

The expression of BDNF and related neurotrophins in uninjured,non-ischemic adult rodent heart was assessed immunohistochemically. BDNFis expressed by endothelial cells lining muscular arteries andarterioles and a proportion of intramyocardial capillaries (FIG. 1A),whereas the related neurotrophin, NT-3, is most highly expressed bycapillaries in adult rodent heart (FIG. 1B). Previous studies havedemonstrated that NT-4 mRNA is expressed at significant levels in theembryonic rat heart but falls to undetectable levels in the adult rodentheart (Timmusk et al., “Widespread and Developmentally RegulatedExpression of Neurotrophin-4 mRNA in Rat Brain and Peripheral Tissues,”Eur. J. Neurosci 5:605-613 (1993), which is hereby incorporated byreference), and are consistent with an inability to detect NT-4 proteinin adult rodent heart sections (FIG. 1C). In the late gestational rodentheart, BDNF and the kinase active isoform of trk B are localized tointramyocardial vessels (FIGS. 1D, E, and control FIG. 1F). Using doubleimmunofluorescence and confocal microscopy, BDNF and trk B co-localizewith the endothelial marker, CD 31 (PECAM), suggesting that endothelialcells express both receptor and ligand in the adult heart (FIGS. 1G andH) and in late gestation at embryonic day E18.5 (FIGS. 1I and J). Thesimilar patterns of expression of BDNF and trk B with CD 31 suggeststhat BDNF and trk B co-localize in the developing intramyocardialarteries in late gestation, and expression of this ligand:receptorsystem by intramyocardial vessels persists into adulthood.

Example 9 Vascular Defects in BDNF (−/−) Mice

To determine whether BDNF performs a critical role in mammalian cardiacor vascular development, the hearts of mice with targeted deletion ofthe BDNF gene were examined. BDNF null mutant (−/−) mice exhibit wellcharacterized losses in trk B expressing peripheral sensory neuronsregulating respiratory rhythm, and in Purkinje neurons (Jones et al.,“Targeted Disruption of the BDNF Gene Perturbs Brain and Sensory NeuronDevelopment but not Motor Neuron Development,” Cell 76:989-999 (1994);Ernfors et al., “Mice Lacking Brain-Derived Neurotrophic Factor Developwith Sensory Deficits,” Nature 368:147-150 (1994); Erickson et al.,“Mice Lacking Brain-Derived Neurotrophic Factor Exhibit Visceral SensoryNeuron Losses Distinct from Mice Lacking NT4 and Display a SevereDevelopmental Deficit in Control of Breathing,” J. Neurosci 16:5361-5371(1996); Schwartz et al., “Abnormal Cerebellar Development and Foliationin the BDNF (−/−) Mice Reveals a Role for Neurotrophins in CNSPatterning,” Neuron 19:269-281 (1997), which is hereby incorporated byreference). The majority of BDNF (−/−) pups die within 1-4 days ofbirth, although approximately 10% of the animals survive for one to twoweeks, with markedly reduced body weight, and impaired spontaneousmovement, a phenotype suggesting potential defects in cardiovasculardevelopment. Upon gross examination of BDNF (−/−) mice at postnatal day0 (P0), the heart size and anatomical relationships of the heart andgreat vessels appeared unremarkable. 14 of 15 BDNF (−/−) animalsexamined at P0, however, exhibited intramyocardial hemorrhage, whichranged from focal areas within the left ventricular wall, to diffusehemorrhage within the walls of both ventricles and in the base of theatria (FIGS. 2B and D, compare to FIGS. 2A and C). Intramyocardialhemorrhage was typically confined to the more epicardial regions of theventricular walls, and was rarely observed in the interventricularseptum.

Because BDNF and trk B are expressed by late gestational and adultintramyocardial vessels, potential defects in vessel morphogenesis inthe BDNF (−/−) animals were assessed by ultrastructural analysis.Abnormalities in the morphology of intramyocardial arterioles (15 of 18vessels examined) were detected in the BDNF (−/−) animals, as comparedto (+/+) littermates (16 vessels examined). These included hypertrophyand abnormal vacuolization of the endothelial cells (15 of 18 vessels),perivascular edema (9 of 18 vessels), and a modest reduction in thenumber of pericytes and vascular smooth muscle cells in the tunica media(10 of 18 vessels) (FIG. 2F, compare to FIG. 2E). Intramyocardialvenules in both the BDNF (−/−) mice (6 vessels examined) and in BDNF(+/+) animals (5 vessels examined) appeared unremarkable (FIG. 2H,compare to FIG. 2G). By electron microscopic analysis, endothelial cellswithin arterioles and in capillaries appeared enlarged and focallydegenerated, with a vacuolated cytoplasm and prominent plasma membraneblebbing (FIGS. 3B and C, compare with FIG. 3A). More than 60% of thecapillary endothelial cells examined (51 of 80) in sections of heartsfrom BDNF (−/−) animals (P0) exhibited cytoplasmic vacuolization,disorganization of the extracellular matrix, and perivascular edema.

To determine whether the ultrastructural changes noted in endothelialcells reflected an apoptotic process, concomitant TUNEL analysis andCD31 immunofluorescence detection was performed using tissue sectionsfrom BDNF (−/−) animals and (+/+) littermates at E18.5 or P2 (FIGS. 3D,E, F, and G). In sections from E18.5 BDNF (−/−) hearts, numerous TUNELpositive cells were detected per low power field and the majority ofthese cells displayed CD31 immunoreactivity, whereas sections from (+/+)embryos exhibited many fewer TUNEL positive cells, which were CD31negative. In examination of P2 BDNF (−/−) hearts, a marked increase inTUNEL positive cells was detected. Although the majority of TUNELpositive cells colocalized with CD31 immunodetection, in regions withhigh TUNEL detection, CD31 negative cells were noted as well, which mayreflect myocyte apoptosis in local regions of vessel compromise. Theseresults suggest that BDNF deprivation results in the apoptosis ofendothelial cells in capillaries and arterioles of the late gestationaland early neonatal heart.

To establish the onset of abnormal intramyocardial vessel formation inthe BDNF (−/−) mice, 11 BDNF (−/−) embryos and 6 BDNF (+/+) littermateswere examined from E11.5 to E19.5. Intramyocardial hemorrhage in BDNF(−/−) embryos could be detected first at E16.5 (2 of 2 BDNF (−/−)embryos from different litters (FIG. 4B)), and was present in 3 of 3BDNF (−/−) embryos (FIG. 4D) examined at E17.5, and absent in BDNF (+/−)and (+/+) littermates (FIGS. 4A and C). The onset of hemorrhage in lategestation suggested that deficient BDNF expression did not impairvasculogenesis or sprouting angiogenesis, as gene targeted embryos withdefects in these processes typically die in utero between embryonic day9-13 (Carmeliet et al., “Abnormal Blood Vessel Development and Lethalityin Embryos Lacking a Single VEGF Allele,” Nature 380:435-439 (1996);Fong et al., “Role of the Flt-1 Receptor Tyrosine Kinase in RegulatingAssembly of Vascular Endothelium,” Nature 376:66-70 (1995); Shalaby etal., “Failure of Blood-Island Formation and Vasculogenesis inFLK-1-Deficient Mice,” Nature 376:62-66 (1995), which are herebyincorporated by reference). Indeed, the density and patterning of theintramyocardial capillary bed in the BDNF (−/−) embryos at E18.5 was notdistinguishable from that of BDNF (+/+) littermates as assessed by CD31immunoreactivity (FIGS. 4G and H). These results suggest that sproutingangiogenesis proceeds normally in the hearts of BDNF (−/−) embryosduring mid to late gestation.

Example 10 Echocardiographic Imaging of BDNF (−/−) Mice

To assess the functional impairment of the BDNF deficient heart in vivo,real time echocardiography was performed on littermates within 48 hoursof birth using a 40 MHz intravascular ultrasound catheter fortransthoracic images. Three of three BDNF (−/−) animals that were imageddisplayed significant decreases in left ventricular ejection fraction(EF) as compared to normal littermates (see Tables 1 and 2, below).

TABLE 1 Ejection fractions for two BDNF (−/−) and three BDNF (−/−)animals from two litters. BDNF (+/+) BDNF (−/−) 73% 42% 77% 62% 31%

TABLE 2 Representative measurements from BDNF (+/+) and BDNF (−/−)littermates. BDNF (+/+) BDNF (−/−) 1vidd 1.10 mm 1.20 mm 1vids 0.70 mm1.00 mm dias vol 1.33 mm³ 1.73 mm³ sys vol 0.34 mm³ 1.00 mm³ strokevolume 0.99 mm³ 0.73 mm³ ejection fraction 73% 42%The reduction in ejection fraction in the BDNF deficient neonates isconsistent with the histologic and ultrastructural evidence ofintramyocardial vessel fragility and hemorrhage which impacts onmyocardial contactility.

Example 11 Deficiency in BDNF Results in Defective Atrial Septation

In addition to vascular defects, microscopic examination of BDNF (−/−)animals was notable for abnormalities in atrial septation in 10 of the12 animals examined by serial section analysis at P0 (FIG. 2B). In theaffected animals, the septum primum appeared to be largely vestigial,while the septum secundum exhibited varying degrees of hypoplasia. Theresult of these defects is incompetence of the foramen ovale with aprominent atrial septal defect involving both the septum primum andsecundum. By morphometric analysis, atrial septal defects in excess of100 microns in the anterior-posterior plane were detected in four of theBDNF (−/−) animals. No atrial septal defects were detected in the 10BDNF (+/−) or (+/+) littermates examined (FIG. 2A). Marked atrialenlargement and atrial wall thinning, with concomitant pulmonarycongestion and occasional intra-alveolar hemorrhage were noted in the 10BDNF (−/−) animals with atrial septal defects. No defects in ventricularseptal formation or in valvulogenesis were detected. Furthermore,ventricular trabeculation and septal muscle formation were unremarkable.

To establish the onset and mechanisms underlying defective atrioseptalformation observed at P0, BDNF (−/−) embryos and (+/+) littermates wereexamined from E11.5 to E18.5 (FIG. 4). The formation of the dorsalcomponent of the septum primum is initiated between E10.5 and E11.5(Kaufman, “The Atlas of Mouse Development,” Academic Press, Inc., SanDiego (1992), which is hereby incorporated by reference, a process whichappears unaffected by deficient BDNF expression (FIGS. 4A and B).However, by E14.5, a stage at which the septum primum has formed, andthe dorsal and ventral ridges of the septum secundum are emerging inwild-type animals, the BDNF (−/−) littermates exhibit hypoplasia of thedeveloping dorsal and ventral components of the septum secundum (FIGS.4C and D). The hypoplasia of the septum secundum in the BDNF (−/−)embryos is progressive at gestational stages E16.5 and E18.5 (FIG. 4F),and results in incompetence of the foramen ovale at P0 (FIG. 2B). Todetermine whether abnormalities in BDNF mediated trk B signaling couldresult in the observed septal hypoplasia, immunohistochemicallocalization of the kinase active trk B and BDNF in the developing atriawas undertaken. Expression of the kinase active trk B receptor and BDNFexpression is detectable in the endocardium of the developing atria, inthe region of the septum primum (FIGS. 4I and J). These results suggestthat BDNF-mediated trk B signaling is required for the persistence andcontinued growth of the atrial septum primum and septum secundum.

Example 12 BDNF Effects on Purified Cardiac Microvascular EndothelialCells

To confirm the direct actions of BDNF on endothelial cells, cultures ofhighly purified neonatal microvascular cardiac endothelial cells wereobtained from wild-type mice. The purity of the cell cultures wasquantitated by flow cytometric analysis of CD31 (PECAM) expression anduptake of DiI-LDL, and was routinely determined to be greater than 95%(FIG. 6A). Using RT-PCR analysis, BDNF mRNA expression was detectable insamples from cardiac microvascular endothelial cells (FIG. 6C),extending recent studies documenting BDNF expression by brainmicrovascular endothelial cells (Leventhal et al., “Endothelial TrophicSupport of Neuronal Production and Recruitment From the Adult MammalianSubependyma,” Mol. Cell. Neurosci. 13:450-464 (1999), which is herebyincorporated by reference). Low passage cardiac microvascularendothelial cells consistently express transcripts for kinase active trkB (FIG. 6C), using well characterized primer sets to detect trk Bisoforms (Labouyrie et al., “Expression of Neurotrophins and TheirReceptors in Human Bone Marrow,” Am. J. Pathol. 154:405-415 (1999),which is hereby incorporated by reference). Transcripts for truncatedtrk B receptors lacking kinase activity were occasionally detected fromRNA obtained from higher passage cells.

Although deficient expression of BDNF in vivo results in endothelialcell apoptosis (as above, FIG. 3E), it is desirable to assess whetherBDNF acts directly upon endothelial cells to support survival underconditions of serum deprivation. Using cultured microvascular cardiacendothelial cells, nanomolar concentration of BDNF was capable ofmaintaining endothelial cell viability, as quantitated using a LIVE/DEADassay. To confirm that BDNF was able to inhibit the endothelial cellapoptosis induced by serum withdrawal, quantitation of apoptosis wasundertaken using TUNEL analysis and annexin V binding (FIGS. 6 D, E, F,and G). Moreover, nanomolar concentrations of BDNF were effective inreducing by approximately fifty percent the cellular apoptosis ofcardiac microvascular endothelial cell followed by serum withdrawal(FIG. 6F, compare with FIG. 6E). BDNF treatment resulted in a 50%reduction in annexin V binding, relative to serum deprived cells (FIG.6G), and was as effective as VEGF in maintaining cell viability. Theability of BDNF to maintain the survival of purified populations ofcardiac microvascular endothelial cells suggests direct actions of thisgrowth factor on this trk B expressing cell population.

Example 13 BDNF Overexpression in the Gestational Heart InducesAngiogenesis

To determine whether excess levels of BDNF may result in vascularabnormalities during embryogenesis, transgenic mice overexpressing BDNFunder the control of the promoter and enhancer regions of the nestingene were generated. These mice used enhancer sequences in intron 1,which direct expression in developing muscle, and sequences in intron 2which are required for expression in the developing nervous system(Zimmerman et al., “Independent Regulatory Elements in the Nestin GeneDirect Transgene Expression to Neural Stem Cells or Muscle,” Neuron12:11-24 (1994), which is hereby incorporated by reference). As thesemice die shortly before birth (Ringstedt et al., “Role of Brain-DerivedNeurotrophic Factor in Target Invasion in the Gustatory System,” J.Neurosci 19:3507-3518 (1999), which is hereby incorporated byreference), transgenic embryos (E 17.5-E18.5) arising from independentinjections of the construct were harvested and found to focallyoverexpress BDNF in the cardiac ventricular walls (FIG. 7F, compare withFIG. 7D). Histologic analysis of the hearts from 6 BDNF overexpressingor 6 wild type embryos revealed focal abnormalities in the ventricularwall of transgenic animals, characterized by an increased number ofpredominantly small diameter vessels (less than 10 microns), whichappeared devoid of a surrounding smooth muscle cell investment (FIG. 7B,compare with FIG. 7A). Immunohistochemistry for the endothelial cellmarker CD31 revealed a 2-3 fold increase in the density of endotheliallined vessels in these regions of the ventricles of transgenic, ascompared to nontransgenic littermates (FIG. 7H, compare with FIG. 7G).No differences in immunopositivity for a-actinin, a vascular smoothmuscle cell marker (Hungerford et al., “Identification of a Novel Markerfor Primordial Smooth Muscle and its Differentiation Expression patternin Contractile vs. Noncontractile Cells,” J. Cell Biol. 137:925-937(1997), which is hereby incorporated by reference), were detected,suggesting that these vessels were capillaries (FIGS. 7I and J). Todetermine whether the increases in vessel number reflected increasedendothelial cell proliferation, immunohistochemical detection ofproliferating cell nuclear antigen (PCNA) was undertaken (FIGS. 7K andL). No differences in PCNA positivity were detected in transgenic, ascompared to wild-type embryos, suggesting that BDNF overexpressionpromotes endothelial cell survival, rather than cell proliferation inviro. Significantly, no evidence of intraventricular wall hemorrhage wasnoted in the BDNF overexpressing embryos suggesting that BDNF does notinduce vascular permeability.

Example 14 Expression of BDNF is Required for the Stabilization ofIntramyocardial Vessels During Late Embryogenesis

The above data demonstrate that expression of BDNF is required for thestabilization of intramyocardial vessels during late embryogenesis,through direct actions on endothelial cells. Unlike well characterizedangiogenic factors, such as VEGF, which initiate vasculogenesis andsprouting angiogenesis, BDNF appears to act at later stages ofarteriolar and capillary formation to maintain vessel integrity. BDNFdoes not appear to regulate vasculogenesis, the patterning of theintra-embryonic vessels, or sprouting angiogenesis, the initialdevelopment of capillaries from these primitive channels, as BDNF (−/−)embryos appear normal through E14.5. Unlike mice deficient in VEGF orthe VEGF receptors flk-1 or flt-1, which die between E8.5 and E11.5 withsevere defects in vasculogenesis, angiogenesis, and yolk sachematopoiesis (Carmeliet et al., “Abnormal Blood Vessel Development andLethality in Embryos Lacking a Single VEGF Allele,” Nature 380:435-439(1996); Fong et al., “Role of the Flt-1 Receptor Tyrosine Kinase inRegulating the Assembly of Vascular Endothelium,” Nature 376:66-70(1995); Shalaby et al., “Failure of Blood Island Formation andVasculogenesis in the Flk-1 Deficient Mice,” Nature 376:62-66 (1995),which is hereby incorporated by reference), BDNF deficient mice displaynormal vascular patterning and capillary branching. The vascularabnormalities in the BDNF deficient mouse are also distinctive fromthose exhibited by animals deficient in expression of angiopoietin-1 andits receptor tyrosine kinase Tie2. Animals lacking expression ofangiopoietin-1 or Tie2 exhibit severe defects in capillary branching,and an inability to remodel the capillary network to form arteries (Satoet al., “Distinct Roles of the Receptor Tyrosine Kinases Tie-1 and Tie-2in Blood Vessel Formation,” Nature 376:70-74 (1995); Suri et al.,“Requisite Role of Angiopoeitin-1, a Ligand for the Tie-2 Receptor,During Embryonic Angiogenesis,” Cell 87:1171-1180 (1996), which ishereby incorporated by reference). Although ultrastructural analysis ofvessels from the angiopoietin-1 and Tie 2 null mutant animalsdemonstrates endothelial cell degeneration, the earlier embryoniclethality of these animals at E10.5-E12, and the widespread vesselabnormalities distinguish the effects of angiopoietin-1 from BDNF.

Recent studies have identified several angiogenic factors which functionto modulate reciprocal interactions between endothelial cells and themesenchymally-derived pericyte and vascular smooth muscle cells. Thesemesenchymal cells are recruited during the process of vascularremodeling and are important in vessel stabilization (Risau, “Mechanismsof Angiogenesis,” Nature 386:671-674 (1997); Darland et al., “BloodVessel Maturation: Vascular Development Comes of Age,” J. Clin. Invest.103:167-168 (1999); Yancopoulos et al., “Vasculogenesis, Angiogenesisand Growth Factors: Ephrins Enter the Fray at the Border,” Cell93:661-664 (1998), which is hereby incorporated by reference). PDGF-BBand HB-EGF synthesized by the endothelial cells recruit pericytes andsmooth muscle cells to the developing tunica media, and deficientPDGF-BB production results in defective vascular ensheathment by thesesupporting cell types (Lindahl et al., “Pericyte Loss and MicroaneurysmFormation in the PDGF-B-deficient mice,” Science 277:242-245 (1997),which is hereby incorporated by reference). The vascular smooth musclecells, in turn, synthesize and secrete angiopoietin-1 to activate Tie2receptors on the adjacent endothelial cells, resulting in bidirectionalsignaling between endothelial cells, and the support cells whichensheath them (Yancopoulos et al., “Vasculogenesis, Angiogenesis andGrowth Factors: Ephrins Enter the Fray at the Border,” Cell 93:661-664(1998), which is hereby incorporated by reference). The co-localizationof BDNF and trk B to endothelial cells of intramyocardial arteries andcapillaries, as well as the ability of BDNF to support the survival ofcardiac microvascular endothelial cells in culture provide mechanisticevidence of direct actions of BDNF on endothelial cells. In addition,ultrastructural analysis of BDNF deficient animals documentingendothelial cell degeneration and apoptosis within intramyocardialcapillaries suggest that this growth factor exerts important roles inendothelial cell survival. The extent of endothelial cell vacuolizationand degeneration observed in the BDNF deficient mice could result indeficient local production of growth factors such as PDGF-BB, thussecondarily impairing smooth muscle cell ensheathment and survival.

The vascular phenotype observed upon BDNF overexpression in thegestational heart further supports the hypothesized role of BDNF as afactor regulating endothelial cell survival and vessel stabilization.Although these animals exhibited increased capillary density, nohemorrhage of these vessels was observed, distinguishing the effects ofBDNF from those of VEGF, which can promote the formation of capillarieswith enhanced fragility. In addition, BDNF overexpression does notsignificantly alter endothelial cell proliferation, suggesting that theincreased capillary density may result from enhanced cell survivalduring the normal processes of vessel remodeling.

The atrial septal defects in the BDNF (−/−) animals, as assessed bymorphometric analysis, are considerably larger than that which has beendescribed for secondary physiologic septal defects. The markedhypoplasia of these septal structures, as well as the local expressionof trk B and the BDNF by the atrial endocardium, suggest that BDNF isrequired for normal septal development in addition to its present rolein maintaining endothelial cell function. This structural defect mayreflect either primary survival deficiencies in the mesenchymal cells ofthe septae or an endothelial cell dysfunction leading to increasedapoptosis of these endothelial/mesenchymal derived structures. There islittle known about the stages of cardiac valvuloseptal development,specifically as to what factors define competent valve formation alongwith appropriate septal development. The overall integrity of thesestructures will most likely be defined by a variety of mechanisms,including the heterogeneity of the endocardial endothelial cell, growthfactors of the TGFβ family, and transcription factors of theHelix-Loop-Helix family (Fishman et al., “Fashioning the VertebrateHeart: Earliest Embryonic Decisions,” Development 124:2099-2117 (1997);Schott et al., “Congenital Heart Disease Caused by Mutations in theTranscription Factor NKX 2.5,” Science 281:108-111 (1998), which ishereby incorporated by reference). Interestingly, the co-existence of acompetent valve system in conjunction with markedly hypoplastic septalstructures in the BDNF−/− animals supports an even more complicatedprofile of pathways that define this period of heart development.

Are there additional actions, however, for BDNF on vascular smoothmuscle cells? Several studies (Nemoto et al., “Gene Expression ofNeurotrophins and Their Receptors in Cultured Rat Vascular Smooth MuscleCells,” Biochem. Biophys. Res. Commun 245:284-288 (1998); Scarisbrick etal., “Coexpression of the mRNAs for NGF, BDNF and NT-3 in theCardiovascular System of Pre and Post Natal Rat,” J. Neurosci.13:875-893 (1993), which is hereby incorporated by reference) havedocumented low levels of expression of BDNF in vascular smooth musclecells from large adult vessels such as the aorta, and prior studies havedocumented increased expression of both BDNF and trk B by neointimalcells following vascular injury (Donovan et al., “Neurotrophin andNeurotrophin Receptors in Vascular Smooth Muscle Cells: Regulation ofExpression in Response to Injury,” A. J. Path. 147:309-324 (1995), whichis hereby incorporated by reference). Migration of medial smooth musclecells is a primary response to vascular injury, and direct chemotacticactions of neurotrophins on the trk receptor expressing adult vascularsmooth muscle cells has been demonstrated. These results suggest thatneurotrophins can mediate direct effects on vascular smooth muscle cellsin adult, large vessels in pathologic models of injury. Althoughoverexpression of BDNF in the developing heart does not lead to enhancedarteriolar formation, or abnormal intramyocardial vessel ensheathment,further studies will be needed to determine whether BDNF can mediatedirect chemotactic or survival effects on pericytes or vascular smoothmuscle cells in other vascular beds.

Although endothelial cells line vessels in all organs, local expressionof growth factors which regulate endothelial cell function can conferspecialization and functional heterogeneity on distinctive great vesselsand the vascular beds (Edelberg et al., PDGF Mediates CardiacMicrovascular Communication,” J. Clin. Invest. 102:837-843 (1998), whichis hereby incorporated by reference). The above analysis of the BDNF(−/−) mice reveals gross hemorrhage only in the heart and lungs, withnormal development of the great vessels, and of vessels within otherorgans such as the kidney and brain. Two distinct mechanisms couldresult in the limited hemorrhage in BDNF (−/−) animals: (1) expressionof an alternate trk B ligand or (2) restricted, regional expression ofBDNF and trk B in the developing embryo. Although BDNF is a selectiveand specific ligand for trk B, an alternative ligand, NT-4, is widelyexpressed during embryogenesis and in the adult (Timmusk et al.,“Widespread and Developmentally Regulated Expression of Neurotrophin-4mRNA in Rat Brain and Peripheral Tissues,” Eur. J. Neurosci. 5:605-613(1993), which is hereby incorporated by reference). In the nervoussystem, studies of BDNF, NT-4, and BDNF/NT-4 double null mutant micesuggest that the functions of these ligands during the development ofthe peripheral nervous system are partially overlapping (Jones et al.,“Targeted Disruption of the BDNF Gene Perturbs Brain and Sensory NeuronDevelopment But Not Motor Neuron Development,” Cell 76:989-999 (1994);Ernfors et al., “Mice Lacking Brain-Derived Neurotrophic Factor Developwith Sensory Deficits,” Nature 368:147-150 (1994); Erickson et al.,“Mice Lacking Brain-Derived Neurotrophic Factor Exhibit Visceral SensoryNeuron Losses Distinct From Mice Lacking NT4 and Display a SevereDevelopmental Deficit in Control of Breathing,” J. Neurosci.16:5361-5371 (1996), which is hereby incorporated by reference). Duringgestation, most non-neuronal tissues express both BDNF mRNA and NT-4mRNA although local expression of both trk B ligands is largelyreciprocal in the adult (Timmusk et al., “Widespread and DevelopmentallyRegulated Expression of Neurotrophin-4 mRNA in Rat Brain and PeripheralTissues,” Eur. J. Neurosci. 5:605-613 (1993), which is herebyincorporated by reference). The developing heart, in contrast, expressespredominately NT-4 mRNA until late gestation, when BDNF mRNA expressionincreases significantly. Thus, the vascular defects in BDNF (−/−)animals may be limited to those organs which express one ligandselectively. Surprisingly, no abnormalities were detected in thedeveloping hearts of NT-4 deficient mice. However, prior studies havedemonstrated that circulating platelets express high levels of BDNF(Yamamoto et al., “Human Platelets Contain Brain-Derived NeurotrophicFactor,” J. Neurosci 10:3469-3478 (1990), which is hereby incorporatedby reference), which may be sufficient to maintain endothelial cellintegrity and survival.

Alternatively, the relatively restricted pattern of BDNF and trk Bexpression to endothelial cells lining some capillaries andintramyocardial arterioles, and the partially overlapping expression ofNT-3 by capillary endothelial cells, suggests that arteriolar andcapillary endothelial cells may require distinct, but related growthfactors to ensure cell survival. The recently described heterogeneity ofcardiac microvascular endothelial cell responsiveness to PDGF-AB, andthe ability of PDGF-AB to selectively regulate endothelial cell geneexpression, suggests that local expression of selective growth factorscan regulate microvascular endothelial cell function (Edelberg et al.,(PDGF Mediates Cardiac Microvascular Communication,” J. Clin. Invest.102:837-843 (1998), which is hereby incorporated by reference). Inaddition, the recent identification of ephrin B2 and its receptor Eph B4as embryonic markers for endothelial cells within arterial or venouscapillaries, respectively, suggests that endothelial cells aremolecularly distinct prior to their ensheathment by vascular smoothmuscle cells (Wang et al., “Molecular Distinction and AngiogenicInteraction Between Embryonic Arteries and Veins Revealed by Ephrin-B2and its Receptor Eph-B4,” Cell 93:741-753 (1998), which is herebyincorporated by reference). One hypothesis to account for the patternsof expression of the neurotrophins by vascular endothelial cells is thatproduction of the neurotrophins may be regulated by ephrin-B2:Eph-B4, orPDGF-AB mediated inter-endothelial signaling, questions most amenable togenetic dissection.

Example 15 Angiogenesis by the trk Receptor Ligands

To assess the potential actions of trk receptor ligands in initiatingangiogenesis in non-ischemic tissues, a well established, non-ischemicin vivo Matrigel model system was utilized (Passaniti et al., “A SimpleQuantitative Method for Assessing Angiogenesis and Antiangiogenic AgentsUsing Reconstituted Basement Membrane, Heparin, and Fibroblast GrowthFactor,” Lab. Invest., 67:519-528 (1992), which is hereby incorporatedby reference). Young adult female mice were injected subcutaneously with0.3 ml of growth factor depleted Matrigel (Bector Dickenson, Bedford,Mass.) containing 64 U/ml heparin and either rhVEGF (UpstateBiotechnologies, Lake Placid, N.Y.) (30-50 ng/ml), rhBDNF (50-100ng/ml), rhNT-4 (50-100 ng/ml), or rhNT-3 (50-100 ng/ml), or no growthfactor addition (rhBDNF, rhNT-4, and rhNT-3 from Promega, Madison,Wis.). After 14 days, the animals were sacrificed, and the Matrigel plugisolated, photographed, and processed for immunohistochemical andhistochemical analysis (see FIGS. 8-10). Serial sections were analyzedin a blinded manner for each Matrigel plug, and the degree ofcellularity was quantitated in central regions of Matrigel. Seven ofeight Matrigel samples containing no additional growth factors exhibitedlow cellularity. In contrast, Matrigel plugs containing VEGF were highlycellular in ten of twelve animals. Matrigel containing trk ligands alsoyielded highly cellular sections; specifically, Matrigel containing BDNFgave rise to highly cellular sections in seven of nine animals, Matrigelcontaining NT-4 was highly cellular in eight of nine animals, andMatrigel containing NT-3 was highly cellular in five of seven animals.

Sections from Matrigel containing VEGF, BDNF, NT-3, NT-4, or noadditional growth factor were examined from 45 animals. Microscopicallythe control Matrigel plug experiments yielded absent to few numbers ofinfiltrating endothelial cells admixed with some scattered mesenchymalcells. By comparison, the Matrigel sections containing VEGF exhibited aloose, fairly organized capillary network which in some cases wascomposed of a dense network of endothelial type cells with focalhemorrhage. In some VEGF-containing Matrigel plugs in which theendothelial/cellular content was rather dense, there were dilated, bloodfilled spaces apparently lined by endothelial cells. When examining theMatrigel plugs containing either the BDNF or NT-4 there was a dramaticincrease in the number of infiltrating endothelial-like cells arborizingthroughout the material when compared with the controls or VEGF treatedsamples. In some of these cases, the dense network of cells wascontained many mitotic figures and apoptotic bodies. Furthermore, insome cases, there were dilated blood filled spaces similar to thosenoted with the VEGF treated plugs. The NT-3 containing Matrigel samplesexhibited a cellular content that was less than that exhibited by theBDNF or NT-4 Matrigel samples but was more dense when compared with theVEGF-Matrigel treated group. In all treated matrigel experiments, therewere varying degrees of an inflammatory component; in the BDNF and NT-4samples, this appeared to be composed of granulocytes includingeosinophils.

Although the present invention has been described in detail for thepurpose of illustration, it is understood that such detail is solely forthat purpose and variations can be made by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. A method of increasing capillary density at aselected site in a subject, said method comprising: selecting a subjectin need of increased vascular density in a site selected from the groupconsisting of the eye, the kidney, and the skin, and administering tothe site of the subject a trk receptor ligand in an amount effective toincrease capillary density at the site, said trk receptor ligand beingselected from the group consisting of brain-derived neurotrophic factor,NT-3, and NT-4.
 2. The method according to claim 1, wherein the subjecthas a condition treatable by increasing capillary density.
 3. The methodaccording to claim 1, wherein the trk receptor ligand is administereddirectly to the site.
 4. The method according to claim 1, wherein thetrk receptor ligand is administered in a manner which targets it to thesite.
 5. The method according to claim 1, wherein the subject has acondition selected from the group consisting of pathological disorders,grafts, transplants, apoptosis, necrosis, and non-cardiac vasculardisorders.
 6. The method according to claim 1, wherein the subject has acondition selected from the group consisting of renal vascular disease,wounds, and proliferative retinopathy.
 7. The method according to claim6, where the subject has a wound selected from the group consisting ofchronic stasis ulcers, diabetic complications, complications of sicklecell disease, thalassemia, disorders of hemoglobin, and post-surgicalwounds.
 8. A method of maintaining the viability of microvascularendothelial cells at a selected site in a subject, said methodcomprising: selecting a subject in need of maintaining viability ofmicrovascular endothelial cells at a selected site, wherein the selectedsite is selected from the group consisting of the eye, the kidney, andthe skin, and administering to the site of the subject a trk receptorligand in an amount effective to maintain the viability of themicrovascular endothelial cells at the site in the subject, said trkreceptor ligand being selected from the group consisting ofbrain-derived neurotrophic factor, NT-3, and NT-4.
 9. The methodaccording to claim 8, wherein the subject has a condition treatable bymaintaining the viability of microvascular endothelial cells.
 10. Themethod according to claim 8, wherein the trk receptor ligand isadministered directly to the site.
 11. The method according to claim 8,wherein the trk receptor ligand is administered in a manner whichtargets it to the site.
 12. The method according to claim 8, wherein thesubject has a condition selected from the group consisting ofpathological disorders, grafts, transplants, apoptosis, necrosis, andnon-cardiac vascular disorders.
 13. The method according to claim 8,wherein the subject has a condition selected from the group consistingof renal vascular disease, wounds, and proliferative retinopathy. 14.The method according to claim 13, where the subject has a wound selectedfrom the group consisting of chronic stasis ulcers, diabeticcomplications, complications of sickle cell disease, thalassemia,disorders of hemoglobin, and post-surgical wounds.